Continuous cell culture system

ABSTRACT

A cell culture system and apparatus is provided for the suspension culturing of mammalian cells in which fresh media can be added and spent medium filtered and withdrawn on a continuous or semi-continuous basis without cell disruption. The apparatus comprises a hollow flask assembly having a hollow shaft means journaled for rotation in a stationary sleeve sub-assembly and a filter unit suspended downwardly therefrom, said filter unit being in fluid communication with the top opening of the flask through the interior of said hollow shaft means and adapted for coaxial rotation with said shaft means.

This is a division of application Ser. No. 850,987, filed Nov. 14, 1977.

BACKGROUND OF THE INVENTION

This invention relates to a continuous cell culture system and apparatustherefor. More particularly, this invention relates to a flask assemblywhich can be used for suspension culturing of mammalian cells in whichfresh medium can be added and spent medium can be separated from thegrowing cells by filtration and withdrawn from the flask on a continuousor semi-continuous basis.

In recent years there has been rapid growth in the development ofvarious methods for the culturing of mammalian cells in suspension. Theattainment of high cell densities is a primary objective of many ofthese approaches. The use of a cell culture vessel with controlledagitation by means of a magnetic stirrer bar or a mechanically drivenimpeller on a shaft is a typical feature of these methods. Examples ofsuch apparatus are disclosed in U.S. Pat. Nos. 2,958,517; 3,039,932;3,572,651; 3,622,129; and 3,649,465. These are essentially batch typespin culture devices in which the cells are incubated in a fixed amountof nutrient under appropriate culture conditions until cell growth hasceased.

It has been recognized that maintenance of constant levels of requirednutrients coupled with removal of toxic cell by-products facilitates thepropagation of cells in higher densities than is obtained in batchprocesses where the cells are grown in a fixed amount of nutrient andharvested after growth ceases. One approach to obtain such higher celldensities employs the batch type apparatus but involves dailycentrifugation and resuspension of cells in fresh medium as reported byClines et al, J. Cell Physiol. 79, 79-90 (1971). Another approach makesuse of special apparatus developed for continuous suspension cellculturing. Examples of such apparatus developed for continuoussuspension cell culturing are disclosed in U.S. Pat. No. 3,647,632; byHimmelfarb, Science 164, 555-57 (1969); and by Thayer et al, "TissueCulture Methods and Applications" (Kruse and Patterson, editors),Academic Press, p. 345-51 (1973). The use of such devices in continuouscell culturing of various cell lines is further described by Thayer etal, Cancer Res. 30, 1709-14 (1970); Cook et al, In Vitro 9, 318-22(1974); and by Lynn and Acton, Biotech. Bioeng. 27, 659-73 (1975).

Notwithstanding the advantages obtained with spin culture devices of theforegoing type, a problem which frequently exists is the presence ofrotating bearing and seal surfaces which can contact the cells in thefluid suspension being agitated and thereby cause grinding and celldisruption at high cell densities. Placement of the rotating shaftdriving means outside the culture vessel in order to avoid the grindingof cells in the flask introduces another problem, namely contaminationby seepage into the vessel around the rotating shaft.

BRIEF SUMMARY OF THE INVENTION

In accordance with the present invention, an improved cell culturesystem and apparatus is provided for the suspension. culturing of cells.This system comprises culturing in a flask assembly in which freshmedium can be added and spent medium can be separated from the growingcells by filtration and withdrawn from the flask on a continuous orsemi-continuous basis. In this system and apparatus, no moving bearingor seal surfaces are present in the culture fluid which could contactthe cells and thereby cause cell disruption, and no rotating shaft isextended through the top of the flask which could permit contaminationfrom the outside to enter the flask during rotation of the shaft.

The apparatus of this invention thus comprises:

(A) a hollow flask having an opening at the top,

(B) an elongated hollow shaft means journaled for axial rotation in astationary sleeve bearing assembly positioned within said flask, influid communication with said top opening and suspended downwardly fromthe top thereof,

(C) a self-supporting filter unit suspended downwardly from said shaftmeans and adapted for coaxial rotation therewith, said filter having

(1) a fluid collection cavity in fluid communication with the interiorof said shaft means and the top opening of said flask, and

(2) a porous peripheral surface having a pore size smaller than thecells to be cultured or the carrier particles upon which said cells areattached.

DETAILED DESCRIPTION OF THE INVENTION

While the specification concludes with claims particularly pointing outand distinctly claiming the subject matter regarded as forming thepresent invention, it is believed that the invention will be betterunderstood from the following description taken in connection with theaccompanying drawings in which:

FIG. 1 is a side elevation view partly in cross section showing anembodiment of the flask assembly of the present invention.

FIG. 2 is an enlarged said elevation view of the rotating filter unit ofFIG. 1 suspended downwardly from the sleeve sub-assembly and tubeconnector means at the top.

FIG. 3 is an exploded view of the sleeve sub-assembly of FIGS. 1 and 2showing partial withdrawal of the hollow shaft means.

FIG. 4 is a side elevation view partly in cross section showing anotherembodiment of the rotating filter unit of the invention.

FIG. 5 is an exploded view of another embodiment of the sleevesub-assembly of the invention.

Now with particular reference to FIGS. 1 and 2, reference numeral 10refers generally to a flask which can be used for the continuoussuspension culturing of mammalian cells. The flask preferably is made ofclear glass or non-toxic rigid plastic materials but also can be made ofbiocompatible metals such as, for example, stainless steel. The flask isshown to have a generally cylindrical form with sidewalls 11, bottom 12,neck portion 13 and mouth 14. It will be appreciated, however, thatother configurations of the flask can be employed. In FIG. 1, the mouthis shown to be closed with a removable stopper 15.

Flask 10 is shown be partially filled with culture fluid 16 in which thecells 17 are suspended. Tubes 18 and/or 19, which pass through holes instopper 15 and thence into the interior of the flask, lead outwardly toa source of culture medium (not shown) which can be supplied to theflask on a continuous or semi-continuous basis. These tubes, or similarsuch tubes, also can be used for withdrawal of cells and cell culturefluid by vertical extension below the level of the culture fluid.

Positioned vertically within the flask is a rotatable filter unit 20which is suspended downwardly from a stationary sleeve sub-assembly 21.The sleeve sub-assembly is held in stopper 15 by the tip portion 23 ofconnector 22 which leads to conduit 24 and thence to a fluid collectionreservoir (not shown). Connector 22 can be, for example, a conventionalSwagelok® O-seal connector which also can be joined to an adaptor, asshown, to accommodate any specific diameter conduit 24.

Filter unit 20 is shown to be held in the sleeve sub-assembly 21 byattachment to an elongated hollow shaft 25 (seen in greater detail inFIG. 3) which, in turn, is journaled for rotation in the sleevesub-assembly. Attachment of the filter unit and the sleeve sub-assemblyis made by a threaded engagement of shaft 25 and a conduit 26. Otherconventional types of fluid-tight fastening means also can be used forthis attachment.

Filter unit 20 is shown to have a generally cylindrical body 27 whichterminates with a rounded bottom and has an internal fluid collectioncavity 28 and an opening in the top 29. The filter unit body can be madeof microporous porcelain, sintered stainless steel, Teflon® plastic orother such microporous filter materials which are fabricated to besufficiently rigid to withstand rotation in use of the apparatus. Forexample, rotation may be at about 100 to 300 rpm during operation of theculture system. In FIG. 2 the thickness of the porous filter wall isshown partly in cross section at the bottom and exaggerated foremphasis.

The pore size of the filter should be smaller than the cells to befiltered, or the carrier particles upon which the cells are attached. Incertain instances it is desired to filter all the cells while in othercases it is desired to filter only the attached cells. A pore size offrom about 0.2 μ to about 7.0 μ is suitable for most single cells. Inthe case of cell aggregates or cells attached to microcarriers, the poresize can be larger but still smaller than the particles, for example, apore size of about 25 μ to 75 μ in the case of particles of about 100 μin diameter. The carrier particles can be materials such as Sephadex®type ion exchange beads, silica glass particles and the like substanceswhich are known to be useful for cell attachment in the suspensionculturing of mammalian cells.

In a 3- or 4- liter size cell culture flask, the commercially availableSelas Flotronics® detachable metal-connector type porcelain filtercandle FDM-126 S can be conveniently used as filter unit 20 with filterconduit 26.

FIG. 3 shows the sleeve sub-assembly in greater detail. Thissub-assembly comprises an upper part 30, a lower part 31 and a centerpart 32. These three parts, which comprise the sleeve sub-assembly, areshown to be generally cylindrical with a centrally disposed borethroughout the entire length. They are adapted for coupling together bythreaded appendages 33 and 34 of the center sleeve part with threadedopenings 35 and 36, respectively, of the upper and lower sleeve parts.Threaded opening 40 of upper sleeve part 30 is adapted for coupling to athreaded shank on connector 22. The bore of the center and lower sleeveparts is adapted for placement therein of the hollow shaft means 25which is shown to be partially withdrawn in FIG. 3. Shaft 25 is seen tobe provided with a flanged top 37 which is adapted for holding the shaftin the sleeve as it is seated on the top of center sleeve part 32. Uppersleeve part 30 preferably has an enlarged opening within its confinessufficient to accomodate the flanged top of shaft 25 (shown more clearlyin the embodiment of FIG. 5). Shaft 25 is threaded internally at thelower end of the shank to provide a fastening means for engagement withthe outer threaded portion of conduit 26 of the filter unit. ElastomericO-ring seal 38 is positioned on the top appendage of the center sleevepart while a similar such seal 39 is positioned on the shank of shaft25. These seals are adapted to prevent fluid leakage as the sleeve partsare held together as shown in FIGS. 1 and 2. Shaft 25 is thus adapted torotate coaxially with filter unit 20 within the stationary sleevesub-assembly 21.

Use of Teflon® plastic materials for the fabrication of the shaft andsleeve sub-assembly parts or plastic coated steel parts providessuitable rotating seal surfaces which require no lubrication.

In another embodiment of the invention shown in FIGS. 4 and 5, variousmodifications are incorporated in the filter unit and sleevesub-assembly of the invention to provide greater rigidity and strengthfor large scale cell culture apparatus. In FIG. 4, filter unit 41 has agenerally cylindrical body 42, an internal fluid collection cavity 43and an opening in the top 44. Positioned within the fluid collectioncavity are a plurality of reinforcement rods 45 which are suspendeddownwardly from an upper disc-shaped plate 46. These rods can be made ofstainless steel or other such suitably rigid materials which arenon-toxic to the fluid cellular product collected in the filter cavity.They are preferably welded onto plate 46 and extend to substantially theentire depth of cavity 43 as shown. In a preferred embodiment of theinvention, four of these rods are employed in the filter unit which areequidistantly spaced apart circumferentially for appropriate balancingof the unit.

Plate 46 has a threaded opening at the center for engagement with thethreaded shank of conduit 47. Conduit 47 is shown to be inserted throughan elastomeric stopper 48 which is squeezed into the lip 49 of thefilter opening as the conduit is threaded into plate 46 to provide anessentially leak-proof enclosure.

The cylindrical filter unit is closed at the bottom by a first bottomplate 50 and a second bottom plate 51. Plate 50 seals against the filterbody 42 by an elastomeric O-ring seal 52 while plate 51 seals againstthe rods 45 by elastomeric O-ring seals 53. Plates 50 and 51 can betightened into a sealing position by engagement of nuts 54 on thethreaded ends of rods 45.

A third bottom plate 55 is positioned below the ends of rods 45 to serveas an attachment means for magnets (not shown). Plate 55 is shown to befastened to plate 51 by screws 56. Small holes 57 drilled through plate55 are adapted for removably wiring the magnets to the bottom of thefilter unit.

It will be appreciated, of course, that other conventional fasteningmeans can be used in place of those illustrated in FIG. 4.

In FIG. 5, the sleeve sub-assembly 59 comprises an upper part 60, alower part 61, a center part 62, and a sleeve insert 63. These fourparts, which comprise the sleeve sub-assembly, are shown to be generallycylindrical with a centrally disposed bore throughout the entire length.Parts 60, 61 and 62 are adapted for coupling together by threaded ends64 and 65 of the center sleeve part with the threaded openings 66 and67, respectively, of the upper and lower sleeve parts. Threaded opening68 of upper sleeve part 60 is adapted for coupling to the threaded shankof a connector which can be, for example, similar to connector 22 inFIG. 1.

Sleeve insert 63 is adapted to frictionally engage the inner walls ofcenter part 62 of the sleeve sub-assembly. The bore of this sleeve isadapted for placement therein of a hollow shaft means 69 which is shownto be partially withdrawn from the center sleeve part in FIG. 5. Shaft69 is seen to be provided with a flanged top 70 which is adapted forholding the shaft in the sleeve as it is seated on the top rim of centersleeve part 62. Upper sleeve part 60 has an enlarged opening within itsconfines sufficient to accommodate the flanged top of shaft 69. Shaft 69is threaded internally at the lower end of the shank to provide afastening means for engagement of the upper threaded portion of conduit47 of the filter unit. Elastomeric O-ring seal 71 is positioned at theupper inside wall of center sleeve part 62 and serves the same functionas seal 39 in the embodiment of FIG. 3. A similar such seal 72 ispositioned below the threaded portion at the upper outside wall of thecenter sleeve part while another similar such seal 73 is positionedunder the foot of the center sleeve part. These three seals are adaptedto prevent fluid leakage when the sleeve parts are sealingly assembledtogether in the manner shown in FIGS. 1 and 2. Shaft 69 is thus adaptedto rotate coaxially with the filter unit 41 within the stationary sleevesub-assembly 59.

It will be appreciated that additional O-ring seals can be employed inthe apparatus of this invention, if desired. For example, a plurality ofO-ring seals interspersed with annular rigid spacers can be positionedat the upper inside wall of center sleeve part 62 instead of the singleO-ring seal 71.

Use of Teflon® and Teflon glass filled plastic materials for thefabrication of the shaft and sleeve insert parts is preferred to providesuitable rotating seal surfaces which require no lubrication. In orderto provide desired additional strength for large scale apparatus, thesleeve subassembly parts 60, 61 and 62 are preferably fabricated fromstainless steel or other such suitably rigid materials which arenon-toxic to cells and cell culture materials employed in operation ofthe apparatus.

When using a Teflon shaft and stainless steel sleeve parts, it ispreferred to additionally employ a steel thrust bearing 74 on theunderside of the flanged top of shaft 69 as shown to hold the weight ofthe filter unit suspended from the shaft. This bearing can be lubricatedwith a high-temperature silicone grease that is non-toxic to any cellsthat may come into contact with the grease. When the apparatus isprovided with such a thrust bearing, a protective hollow tube also canbe positioned within the bore of the sleeve assembly to prevent anyfluid which rises during fluid withdrawal through the filter unit fromcontacting the bearing. For example, a glass tube 76 can be frictionallysuspended from the inside of a connector such as connector 22 of FIGS. 1and 2 and extended downwardly into the center sleeve part 62, preferablyto about 1/4 to 1/2 the depth of the center sleeve part.

In operation of the cell culture apparatus of this invention, using theembodiment of FIG. 1 for illustrative purposes, an inoculum of cells anda suitable culture medium are incubated in flask 10 under cell cultureconditions which are appropriate for the particular cells used. Tubes 18and 19 can be used to supply fresh culture medium to the flaskcontinuously or at predetermined intervals. Magnetic bars (not shown)can be removably attached to the bottom of the outside of the filterunit which can then be caused to rotate by a revolving U-shaped magnet(not shown) positioned under the cell culture flask. The filter unit ispreferably suspended to a depth in the flask such that it liescompletely in the fluid medium. A vacuum drawn on line 24 by aperistaltic pump (not shown), which line is in direct fluidcommunication with the interior of the filter unit through theintermediately disposed hollow shaft means and sleeve sub-assembly, willcause withdrawal of fluid medium from the flask after passage throughthe microporous filter. Culture fluid can thus be separated from theresidual cells on a continuous or semi-continuous basis. The freshculture medium which is added through tubes 18 and 19 also can bemonitored by a level controller (not shown) to maintain a constant levelof suspension in the flask, if desired.

Cell culture apparatus of the present invention as described hereineliminates the usual need for a lower bearing surface in the flask belowthe upper level of the culture fluid. There are no moving bearing orseal surfaces in the flask assembly in contact with the suspensionculture which could cause cell disruption at high densities. The filterunit is self-supporting and there are no moving seals between the filterunit and any filter support means which could contact the cellsuspension. All seals within the filter unit are static. As no lowerbearing is used, a centrally positioned solid shaft is not requiredwithin the filter unit and flow of effluent is not limited to an annularregion around such shaft.

The cell culture apparatus of this invention can be used withconventional auxiliary apparatus such as pumping means for pumping infresh medium from a supply reservoir and pumping out spent media,product, and toxic materials into an effluent collection reservoir. Theflask can be provided with additional openings for gas inlet and outlet,sampling tubes and pH and liquid level monitoring probes. The entireflask assembly should be operated under sterile conditions throughoutthe cell growth period.

The cell culture apparatus of this invention also can be operated as asatellite flask in a series with other cell culture reactors by suitabletubing and pumping means. For example the present apparatus can serve asa filtering means for a main cell culture flask used for the growth ofthe cells.

In illustrative examples of the present invention using a 4-litercapacity cell culture flask in accordance with the apparatus of FIGS. 1to 3 described hereinbefore, in 2 cell culture runs for over 100 hourseach at 250 rpm, the runs were completed without any problems associatedwith cell disruption. Walker 256 Carcinosarcoma cells (ATCC No. CCL 38)converted to suspension growth were thus grown in Dulbecco's ModifiedMinimum Essential Medium (with 4.5 mg/ml of glucose) supplemented with7% fetal calf serum. The cells were seeded at less than 10⁶ cells per mland reached a maximum of 3× 10⁷ cells per ml with doubling times of 14to 18 hours. More than 30 liters of media were passed through the filterin each run. Cells were healthy with high viability through long periodsof log phase growth (up to 85 hours). These cells are useful for theproduction of tumor angiogenesis factor as is known from the publicationby Folkman and Klagsbrun in Chapter 31 of "Fundamental Aspects ofNeoplasia", at pages 401-412, edited by Gottlieb et al, Springer-Verlag,New York, 1975.

In further such illustrative cell culture runs using a 40-liter capacitycell culture flask provided with the filter unit and sleeve sub-assemblyembodiments of FIGS. 4 and 5, the cells reached a maximum of 1.4× 10⁷cells per ml in 120 hours without any problems associated with celldisruption.

In still another illustrative run, the 40-liter flask was operated as achemostat in which the cell density was continuously held at a level offrom 6× 10⁶ to 10× 10⁶ cells per ml during a 4 day culture period and inwhich cells and cell fluid were periodically harvested and fresh mediumwas periodically added as required. Cell fluid was thus withdrawnthrough the filter, and both cells and cell fluid were withdrawnunfiltered through an outlet tube such as tube 18 or 19. Such continuousharvesting permitted more cells and cell fluid product to be harvestedin a given time than under conventional batch cell culture.

It will be appreciated that the continuous cell culture system of thisinvention is adaptable to any of the well-known tissue culture mediasuch as, for example, Basal Medium Eagle's (BME), Eagle's MinimumEssential Medium (MEM), Dulbecco's Modified Eagle Medium, Medium 199,and balanced salt solutions (BSS) such as those of Earle and Hanksfortified with various nutrients. These are commercially availabletissue culture media and are described in detail by H. J. Morton, InVitro 6, 89-108 (1970). These conventional culture media contain knownessential amino acids, mineral salts, vitamins and carbohydrates. Theyare also frequently fortified with mammalian sera such as fetal calfserum.

The present invention also is adaptable to all types of animal cells,including, for example, mammalian, fowl and amphibian cells. Primarycells taken from embryonic, adult or tumor tissues as well as cells ofestablished cell lines can thus be used. Examples of typical such cellsare primary rhesus monkey kidney cells, baby hamster kidney cells, pigkidney cells, embryonic rabbit kidney cells, mouse embryo fibroblasts,normal human lung embryo fibroblasts, HeLa cells, primary and secondarychick fibroblasts, and various cells transformed with SV-40 or polyomavirus.

Growth of these and other such cells in suitable nutrient culture mediaemploying the continuous cell culture system of this invention canthereby be carried out in a manner to provide high cell densitieswithout any moving bearing or seal surfaces contacting the cells whichcould cause cell disruption during the cell culture period.

Various other examples will be apparent to the person skilled in the artafter reading of the disclosure herein without departing from the spiritand scope of the invention. All such further examples are includedwithin the scope of the appended claims.

What is claimed is:
 1. In the method of continuous cell culturecomprising growing cells in an agitated liquid suspension of nutrientculture medium containing known amino acids, mineral salts, vitamins andcarbohydrates and periodically removing filtered liquid from the cells,the improvement comprising carrying out the agitation and filtering inapparatus comprising a hollow flask having an opening at the top, anelongated hollow shaft means and a stationary sleeve assembly positionedwithin said flask, said elongated hollow shaft means journaled for axialrotation in said stationary sleeve assembly, in fluid communication withsaid top opening of said flask and supported downwardly therefromwithout any lower bearing, and a filter unit supported downwardly fromsaid elongated hollow shaft means without any lower bearing and withoutany moving seals between the filter unit and any filter support meanswhich could contact the cell suspension and adapted for coaxial rotationwith said elongated hollow shaft means, said filter having a fluidcollection cavity in direct fluid communication with the interior ofsaid shaft means and the top opening of said flask, and a porousperipheral surface having a pore size smaller than the cells to becultured or the carrier particles upon which said cells are attached butsufficiently large to permit permeation of fluid into said fluidcollection cavity.